Vibrational optical activity: From discovery and development to future challenges.

Vibrational optical activity (VOA) consisting of infrared vibrational circular dichroism (VCD) and vibrational Raman optical activity (ROA) was predicted, discovered, and confirmed between 1971 and 1975. My path to VOA was mentored by three pioneers of chirality and vibrational spectroscopy: Professors Albert Moscowitz, Warner L. Peticolas, and Philip J. Stephens, and while they are no longer alive today, the Chirality Medal, my award address, and this paper are dedicated to each of them. Since the discovery of VOA, a number of key advances have made possible the current era of widespread applications. The principal instrumental advances were Fourier-transform VCD (FT-VCD) and multichannel charge coupled detector (CCD) ROA. Computational advances include the first complete quantum chemistry formulation of VCD leading to the magnetic field perturbation (MFP) and the nuclear velocity perturbation (NVP) theories. The strength of VOA is the comparison between measured and calculated spectra that enables the determination of absolute configuration and solution-state conformations. More recently, VCD has uncovered supramolecular chirality in amyloid fibrils and ROA to high-order protein structure. Future challenges for VOA include describing the effects of weak intermolecular interactions, transfer of chirality, solvent effects, supramolecular chirality, and the generation of nuclear velocity electron current density.

Origin of enhanced VCD in amyloid fibril spectra: Effect of deuteriation and pH.

Supramolecular chirality of amyloid fibrils, protein aggregates related to many neurodegenerative diseases, is a remarkable property associated with fibril structure and polymorphism. Since its discovery almost 10 years ago there is still little understanding of this phenomenon, including the cause of the highly enhanced vibrational circular dichroism (VCD) intensity arising from fibril supramolecular chirality. In this study, VCD spectra, enhanced by filament supramolecular chirality, are presented for lysozyme and insulin fibrils above and below pH 2 and after deuterium exchange, above and below pD 2. Supramolecular chirality (observed by VCD) and fibril morphology (documented by atomic force microscopy) are not affected by protein deuteriation. In D2 O the fibril VCD sign pattern changes to fewer bands, with implications for the amide I/II origin of enhanced VCD intensity. Separation of amide I and II signals will facilitate calculations of enhanced VCD spectra of amyloid fibrils and enable a better understanding of the origin of the VCD sign pattern.

Unravelling a Direct Role for Polysaccharide ß-Strands in the Higher Order Structure of Physical Hydrogels.

The mechanical properties of agarose-derived hydrogels depend on the scaffolding of the polysaccharide network. To identify and quantify such higher order structure, we applied Raman optical activity (ROA)-a spectroscopic technique that is highly sensitive toward carbohydrates-on native agarose and chemically modified agarose in the gel phase for the first time. By spectral global fitting, we isolated features that change as a function of backbone carboxylation (28, 40, 50, 60, 80, and 93 %) from other features that remain unchanged. We assigned these spectral features by comparison to ROA spectra calculated for different oligomer models. We found a 60:40 ratio of double- and single-stranded α-helix in the highly rigid hydrogel of native agarose, while the considerably softer hydrogels made from carboxylated agarose use a scaffold of unpaired ß-strands.

Substrate-Determined Diastereoselectivity in an Enzymatic Carboligation.

Thiamine diphosphate-dependent enzymes catalyze the formation of C-C bonds, thereby generating chiral secondary or tertiary alcohols. By the use of vibrational circular dichroism (VCD) spectroscopy we studied the stereoselectivity of carboligations catalyzed by YerE, a carbohydrate-modifying enzyme from Yersinia pseudotuberculosis. Conversion of the non-physiological substrate (R)-3-methylcyclohexanone led to an R,R-configured tertiary alcohol (diastereomeric ratio (dr) >99:1), whereas the corresponding reaction with the S enantiomer gave the S,S-configured product (dr>99:1). This suggests that YerE-catalyzed carboligations can undergo either an R- or an S-specific pathway. We show that, in this case, the high stereoselectivity of the YerE-catalyzed reaction depends on the substrate's preference to acquire a low-energy conformation.

Three-Dimensional Chemical Structure Search Using the Conformational Code for Organic Molecules (CCOM) Program.

Searching the 3D structural fragments of organic molecules is challenging because of structural differences between X-ray and theoretically calculated geometries and the conformational flexibility of substituents. The codification program called Conformational Code for Organic Molecules (CCOM) can be used to unambiguously convert 3D conformational data for various molecules to 1D data. Two deviations from Rule E-5.6 of the International Union of Pure and Applied Chemistry (IUPAC) Rules for Nomenclature of Organic Chemistry were introduced to the CCOM program for 3D fragment searching. First, the search for the highest priority atom was limited to a distance of two bonds from the center bond for dihedral angle determination. Second, for indistinguishable atoms in experimentally observed solution structures, the smallest number of atom index in the molecular model was chosen as the priority atom for dihedral angle determination. A search of the 3D conformational fragment mb_3a6c4c of mevastatin () in combination with the SMiles ARbitrary Target Specification (SMARTS) description suggested that a change in the conformation of this fragment may be the driving force for dissociation of mevastatin from its target protein. Chirality 28:370-375, 2016. © 2016 Wiley Periodicals, Inc.

The transition from the native to the acid-state characterized by multi-spectroscopy approach: study for the holo-form of bovine α-lactalbumin.

The transition of the holo-form of bovine α-lactalbumin from the native (N) to the pH-generated acidic-state (A-state) was analyzed by probing its tertiary and secondary structure using a concerted spectroscopic approach combining near- and far-UV circular dichroism (CD), electrospray ionization ion mobility mass spectrometry (ESI-IM-MS), vibrational circular dichroism (VCD), and Fourier transform infrared spectroscopy (FTIR) in the attenuated total reflection (ATR) and transmission (TR) modes. The spectroscopic results, which relied on the interaction of an electromagnetic field with different molecular targets, confirmed the decay of extensive rigid side-chain packing interactions during the pH-induced N→A-state transition and revealed the targets' dependence on secondary structural changes. Independent analyses of the spectral changes using two methods of multivariate analysis, such as principal component analysis and two-dimensional correlation spectroscopy, revealed small but significant differences in the secondary structure as a result of the all-or-none transition. The cooperativity of the transition was quantitatively described using values corresponding to the mid-point (tm) and width of the transition (Δtm). The averages of the two parameters, calculated using the data collected by the different probes, were equal to 3.5±0.2 and 0.6±0.1(SE), respectively. The variable two-state nature of the cooperative N→A-state transition confirmed that the protonation of the side chain carboxyl groups on the Asp and Glu residues and that the release of a Ca(2+) ion induced structural changes on both the secondary and tertiary levels. The changes have been confirmed by results obtained from the concerted spectroscopic approach.

Solvation-induced helicity inversion of pseudotetrahedral chiral copper(II) complexes.

The helicity of four-coordinated nonplanar complexes is strongly correlated to the chirality of the ligand. However, the stereochemical induction of either the Δ- or the Λ-configuration at the metal ion is also modulated by environmental factors that change the conformational distribution of ligand rotamers. Calculation of the potential energy surface of bis{(R)-N-(1-(4-X-phenyl)ethyl)salicylaldiminato-κ(2)N,O}copper(II) with X = Cl at the density functional theory level showed a clear dependence of the helicity-determining angle θ between the two coordination planes on the relative population of different ligand conformers. The influence of different substituents (X = H, Cl, Br, and OCH3) on complex helicity was studied by determination of the absolute configuration at the metal ion in complexes with either (R)- or (S)-configured ligands. X-ray single-crystal analysis showed that (R)-configured ligands with H, Cl, Br induce Δ, while OCH3-substituted (R)-configured ligands induce Λ in the solid state. According to vibrational circular dichroism and electronic circular dichroism studies in solution, however, all tested complexes with (R)-ligands exhibited a propensity for Δ, with high diastereomeric ratio for X = Cl and X = Br and moderate diastereomeric ratio for X = H and X = OCH3 substituted ligands. Therefore, solvation of copper complexes with X = OCH3 goes along with helicity inversion. This solid-state versus solution study demonstrates that it is not sufficient to determine the chiral-at-metal configuration of a compound by X-ray crystallography alone, because the solution structure can be different. This is particularly important for the use of chiral-at-metal complexes as catalysts in stereoselective synthesis.

Optically Active Nanostructured ZnO Films.

Inorganic nanomaterials endowed with hierarchical chirality could open new horizons in physical theory and applications because of their fascinating properties. Here, we report chiral ZnO films coated on quartz substrates with a hierarchical nanostructure ranging from atomic to micrometer scale. Three levels of hierarchical chirality exist in the ZnO films: helical ZnO crystalline structures that form primary helically coiled nanoplates, secondary helical stacking of these nanoplates, and tertiary nanoscale circinate aggregates formed by several stacked nanoplates. These films exhibited optical activity (OA) at 380 nm and in the range of 200-800 nm and created circularly polarized luminescence centered at 510 nm and Raman OA at 50-1400 cm(-1) , which was attributed to electronic transitions, scattering, photoluminescent emission, and Raman scattering in a dissymmetric electric field. The unprecedented strong OA could be attributed to multiple light scattering and absorption-enhanced light harvesting in the hierarchical structures.

Is supramolecular filament chirality the underlying cause of major morphology differences in amyloid fibrils?

The unique enhanced sensitivity of vibrational circular dichroism (VCD) to the formation and development of amyloid fibrils in solution is extended to four additional fibril-forming proteins or peptides where it is shown that the sign of the fibril VCD pattern correlates with the sense of supramolecular filament chirality and, without exception, to the dominant fibril morphology as observed in AFM or SEM images. Previously for insulin, it has been demonstrated that the sign of the VCD band pattern from filament chirality can be controlled by adjusting the pH of the incubating solution, above pH 2 for "normal" left-hand-helical filaments and below pH 2 for "reversed" right-hand-helical filaments. From AFM or SEM images, left-helical filaments form multifilament braids of left-twisted fibrils while the right-helical filaments form parallel filament rows of fibrils with a flat tape-like morphology, the two major classes of fibril morphology that from deep UV resonance Raman scattering exhibit the same cross-ß-core secondary structure. Here we investigate whether fibril supramolecular chirality is the underlying cause of the major morphology differences in all amyloid fibrils by showing that the morphology (twisted versus flat) of fibrils of lysozyme, apo-α-lactalbumin, HET-s (218-289) prion, and a short polypeptide fragment of transthyretin, TTR (105-115), directly correlates to their supramolecular chirality as revealed by VCD. The result is strong evidence that the chiral supramolecular organization of filaments is the principal underlying cause of the morphological heterogeneity of amyloid fibrils. Because fibril morphology is linked to cell toxicity, the chirality of amyloid aggregates should be explored in the widely used in vitro models of amyloid-associated diseases.

Amplified vibrational circular dichroism as a probe of local biomolecular structure.

We show that the VCD signal intensities of amino acids and oligopeptides can be enhanced by up to 2 orders of magnitude by coupling them to a paramagnetic metal ion. If the redox state of the metal ion is changed from paramagnetic to diamagnetic the VCD amplification vanishes completely. From this observation and from complementary quantum-chemical calculations we conclude that the observed VCD amplification finds its origin in vibronic coupling with low-lying electronic states. We find that the enhancement factor is strongly mode dependent and that it is determined by the distance between the oscillator and the paramagnetic metal ion. This localized character of the VCD amplification provides a unique tool to specifically probe the local structure surrounding a paramagnetic ion and to zoom in on such local structure within larger biomolecular systems.

Effect of Conformational Variability on the Drug Resistance of Candida auris ERG11p and FKS1.

Candida auris infection has been recognized as an urgent threat to antifungal drug resistance, and the Eagle effect of C. auris FKS1 (1,3-ß-d-glucan synthase) wild-type isolates has also been noted. The Eagle effect, namely, where higher concentrations of antifungals reduce fungicidal activity relative to lower concentrations, is a confounding factor of apparent antifungal resistance, but the detailed mechanism remains unclear. Here, we present the conformational variability of mutation sites for ERG11p (lanosterol 14α-demethylase) and FKS1 from deep neural network-based prediction along with the reported X-ray crystallographic and cryo-electron microscopy (cryo-EM) structures of antifungals. The sequence variability maps provide valuable insights into the inconsistent correlation between azole resistance and the mysterious Eagle effect with the dispersion of minimal inhibitory concentration (MIC) for echinocandin resistance. The conformational variability prediction supports the hypothesis that mutations K143R of clade I, VF125AL of clade III, and Y132F of clade IV for C. auris ERG11p make the corresponding site variable and that an increased population of invisible variable conformations potentially contributes to triazole resistance. In contrast, the predicted rigid conformation by the S639F mutation of hot spot region 1 (HS1) for FKS1 suggests that caspofungin (CAS) is involved in an uncompetitive inhibition, and a decreased population of the CAS-bound state of FKS1 with Rho1 leads to drug resistance. The predicted variable HS1 region for FKS1 WT isolates and the rigid one for FKS1 S639F mutants support the in vivo drug response and the in vitro MIC dispersion. A plausible mechanism of the Eagle effect is hereby proposed, namely, that a high concentration of CAS with a high membrane affinity reduces the population of the CAS-bound state of FKS1 with Rho1, as well as accompanying events such as aggregation or association depending on the conformational variability of HS1.

Data mining of supersecondary structure homology between light chains of immunogloblins and MHC molecules: absence of the common conformational fragment in the human IgM rheumatoid factor.

It is shown that fuzzy search and data mining techniques of supersecondary structure homology for subunits of proteins using conformational code patterns of α-helix-type (3ß5α4ß) and ß-sheet-type (6α4ß4ß) fragments can be used to extract correlations between fragments of MHC class I molecules and the light chain of immunoglobulins. The new method of conformational pattern analysis with fuzzy search of structural code homology reflects well the shape of main chain rather than secondary structure in comparison with the DSSP method. Further, the data mining technique using the combination of h- and s-fragment patterns can quantify the supersecondary structure homology between any subunits of proteins with different amino acid sequences. Characteristic fragment patterns (string "shhshss"), which were sandwiched between two identical amino acid sequences His and Pro, were found in light chains of various types of immunogloblins, α-chain and ß-2 microglobulin of MHC class I and α-chain and ß-chain of MHC class II, but not in heavy chains of Fab immunoglobulin fragments and T cell receptors (TCR). Leukocyte immunoglobulin-like receptors (LILR) are related by the conformational fragment (string "shhshss") to ß-2 microglobulins as a type of pair forms (string "sohsss"). Further, human IgM rheumatoid factor, one of the immunogloblins, did not strongly exhibit the conformational fragment pattern. Nonclassic MHC class I molecules CD1D, MIC-A, and MIC-B, which have functions to activate NKT, NK, and T cells, did not also clearly show the patterns. These code-driven mining techniques can be utilized as a metadata-generating tool for systems biology to elucidate the biological function of such conformational fragments of MHC I and II molecules, which come in contact with various signal ligands on the surface of T cells and natural killer cells.

Exceptionally enhanced Raman optical activity (ROA) of amyloid fibrils and their prefibrillar states.

Amyloid fibrils form remarkable, multi-layered chiral supramolecular architectures. The proximity of interacting oscillators in the chiral fibril supramolecules is responsible for the unusual sensitivity of vibrational circular dichroism (VCD) for fibril formation. Surprisingly, up to now, such characteristics have not been shown for ROA, although it displays the same vibrational markers of fibrils as VCD, including the amide I band. Here, we report an exceptionally large enhancement of the ROA signal detected for mature amyloid fibrils and their prefibrillar states. Remarkably, the same ROA signal has been obtained for fibrils of homologous lysozymes and the dissimilar protein, insulin, indicating a possible common enhanced ROA spectrum, analogous to that for VCD for all amyloid fibrils investigated to date. The ROA signal is observed at earlier stages of fibril formation than VCD and provides access to a considerably broader range of vibrations. Further studies are necessary to verify the applicability of ROA for the analysis of amyloid fibrils.

Effect of Conformational Variability on Seasonable Thermal Stability and Cell Entry of Omicron Variants.

The Omicron BA.1 variant of SARS-CoV-2 preferentially infects through the cathepsin-mediated endocytic pathway, but the mechanism of cell entry has not been solved yet because BA.4/5 is more fusogenic and more efficiently spread in human lung cells than BA.2. It has been unclear why the Omicron spike is inefficiently cleaved in virions compared with Delta, and how the relatively effective reproduction proceeds without the cell entry through plasma membrane fusion. Conformational variability from deep neural network-based prediction correlates well with the thermodynamic stability of variants. The difference of seasonable pandemic variants in summer and those in winter is distinguishable by this conformational stability, and the geographical optimization of variants is also traceable. Further, the predicted conformational variability maps rationalize the less efficient S1/S2 cleavage of Omicron variants and provide a valuable insight into the cell entry through the endocytic pathway. It is concluded that conformational variability prediction is able to complement transformation information on motifs in protein structures for drug discovery.

Normal and reversed supramolecular chirality of insulin fibrils probed by vibrational circular dichroism at the protofilament level of fibril structure.

Fibrils are ß-sheet-rich aggregates that are generally composed of several protofibrils and may adopt variable morphologies, such as twisted ribbons or flat-like sheets. This polymorphism is observed for many different amyloid associated proteins and polypeptides. In a previous study we proposed the existence of another level of amyloid polymorphism, namely, that associated with fibril supramolecular chirality. Two chiral polymorphs of insulin, which can be controllably grown by means of small pH variations, exhibit opposite signs of vibrational circular dichroism (VCD) spectra. Herein, using atomic force microscopy (AFM) and scanning electron microscopy (SEM), we demonstrate that indeed VCD supramolecular chirality is correlated not only by the apparent fibril handedness but also by the sense of supramolecular chirality from a deeper level of chiral organization at the protofilament level of fibril structure. Our microscopic examination indicates that normal VCD fibrils have a left-handed twist, whereas reversed VCD fibrils are flat-like aggregates with no obvious helical twist as imaged by atomic force microscopy or scanning electron microscopy. A scheme is proposed consistent with observed data that features a dynamic equilibrium controlled by pH at the protofilament level between left- and right-twist fibril structures with distinctly different aggregation pathways for left- and right-twisted protofilaments.

Experimental and theoretical polarized Raman linear difference spectroscopy of small molecules with a new alignment method using stretched polyethylene film.

This paper reports the development of the new technique of Raman linear difference (RLD) spectroscopy and its application to small molecules: anthracene and nucleotides adenosine-5'-monophosphate, thymidine-5'-monophosphate, guanosine-5'-monophosphate, and cytidine-5'-monophosphate. In this work we also present a new alignment method for Raman spectroscopy where stretched polyethylene films are used as the matrix. Raman spectra using light polarized along the orientation direction and perpendicular to it are reported. The polyethylene (PE) film spectra are consistent with powder samples and films deposited on quartz. RLD spectra determined from the difference of the parallel and perpendicular polarized light Raman spectra are also reported. The equations describing RLD are derived, and RLD spectra of anthracene and thymidine are calculated from these equations using Density Functional Theory and assuming perfect orientation of the samples. Because of the wealth of spectroscopic information in the vibrational spectra of biomolecules together with our ability to calculate spectra as a function of orientation, we conclude that RLD has the potential to provide structural information for biological samples that currently cannot be extracted from any other method.

VCD to determine absolute configuration of natural product molecules: secolignans from Peperomia blanda.

The absolute configuration and solution-state conformers of three peperomin-type secolignans isolated from Peperomia blanda (Piperaceae) are unambiguously determined by using vibrational circular dichroism (VCD) spectroscopy associated with density functional theory (DFT) calculations. Advantages of VCD over the electronic form of CD for the analysis of diastereomers are also discussed. This work extends our growing knowledge about secondary metabolites within the Piperaceae family species while providing a definitive and straightforward method to assess the absolute stereochemistry of secolignans.

Simultaneous resonance Raman optical activity involving two electronic states.

In the present work, the first observation of strong resonance Raman optical activity (RROA) involving more that one resonant electronic state is reported. The chiral transition metal complex bis-(trifluoroacetylcamphorato) copper(II), abbreviated Cu(tfc)(2), exhibits both resonance Raman (RR) and RROA spectra with laser excitation at 532 nm. Vibrational assignments for this complex were carried out by comparing the non-RR spectra of Cu(tfc)(2) excited at 1024 nm to density functional theory (DFT) calculations. The theory of the single-electronic-state (SES) RROA is extended to the next simplest level of theory involving two resonant electronic states (TES) without interstate vibronic coupling as an aide to the interpretation of the observed TES-RROA spectra. Based on measured UV-vis electronic absorbance spectra and corresponding TD-DFT calculations, the most likely two states associated with the RROA spectra are identified.

A confidence level algorithm for the determination of absolute configuration using vibrational circular dichroism or Raman optical activity.

Spectral comparison is an important part of the assignment of the absolute configuration (AC) by vibrational circular dichroism (VCD), or equally by Raman optical activity (ROA). In order to avoid bias caused by personal interpretation, numerical methods have been developed to compare measured and calculated spectra. Using a neighbourhood similarity measure, the agreement between a computed and measured VCD or ROA spectrum is expressed numerically to introduce a novel confidence level measure. This allows users of vibrational optical activity (VOA) techniques (VCD and ROA) to assess the reliability of their assignment of the AC of a compound. To that end, a database of successful AC determinations is compiled along with neighbourhood similarity values between the experimental spectrum and computed spectra for both enantiomers. For any new AC determination, the neighbourhood similarities between the experimental spectrum and the computed spectra for both enantiomers are projected on the database allowing an interpretation of the reliability of their assignment.